Cryptococcus neoformans (Cn) secretory proteins are believed to be involved in mediating the interaction between the pathogens and host cells during the infection. Cn secretory pathways and machinery involved in the targeting, processing and transport of proteins are largely unexplored. In general, proteins transport to the cell surface follows classical secretory route, i.e. from endoplasmic reticulum to Golgi to plasma membrane, and finally to the cell surface. Interestingly, several physiologically important eukaryotic proteins have been identified that lack a classical signal sequence for secretion, but are still secreted from the cells. Our preliminary data with Cn copper zinc superoxide dismutase (SOD1p) revealed that (a) SOD1p, a cytosolic, single copy, leaderless protein, is secreted outside of the cell through channels in the cell wall, (b) secreted SOD1p is associated with cell wall and capsular material through as yet unknown mechanism, and (c) trapped SOD1p quenches phagocyte oxidative burst more efficiently, and thereby protects Cn from phagocyte killing. Additionally, Cn genomic database searches (Stanford University and The Institute for the Genomic Research) revealed full length CnSTE60RF containing 5121-bp nucleotide with 1706 amino acids, which showed 44% homology to Saccharomyces cerevisiae STE6. S. cerevisiae STE6 mediates export of a lipopeptide mating pheromone a-factor that lacks a classical hydrophobic signal peptide for secretion. Similarly, Cn database searches also revealed 200-300-bp contigs with 50 to 60% homology to nonclassical export genes NCE1, 2, 3 (now named as NCE101, NCE102, NCE103). These genes are well recognized for leaderless protein secretion in S. cerevisiae. These results indicated that Cn possesses nonclassical secretory pathways for protein export. Thus, both classical and nonclassical protein secretory pathways can be studied in Cn to better understand the invasion, survival, multiplication, and dissemination of this pathogen in the host. A number of inter-disciplinary approaches will be used in this investigation based on the previous research training of the principal investigator in Toxoplasma gondii protein trafficking. In specific Aim 1, the nonclassical SOD1p secretion will be compared with classical secretion of capsular protein CAP10p and phospholipase B (PLB1p) using inhibitors of secretion and localization of monoclonal antibodies. In specific Aim 2, two gene regulators of nonclassical secretion (STE6 and NCE102) will be characterized by a combination of molecular genetics and cell biological approaches to determine their relative influence on SOD1p, CAP10p & PLB1p secretions. The results of this investigation are likely to substantially enhance our understanding of protein trafficking in pathogenic fungi in general, and possibly assist in search for new therapeutic targets.